Hyaluronic acid belongs to glycosaminoglycans (GAGs), like heparan sulfate, chondroitin sulfate, dermatan sulfate, and herapin, and is a polyanionic natural linear polymer of repeating units, each composed of N-acetyl-D-glucosamine and D-glucuronic acid. Hyaluronic acid is found in the eyes, the placenta, the synovial fluid lubricant of body joints, the skin, and the comb of chicken and ranges in molecular weight from 103 to 107 Daltons depending on the in vivo site at which it is found. In addition, hyaluronic acid is a main component of the extracellular matrix plays an important role as a scaffold in all layers of the skin including the epidermis and dermis. Hyaluronic acid is also found in the synovial fluid, the umbilical cord, and the blood of all higher animals, and almost 50% of the body's hyaluronic acid is located in the skin, the respiratory tract, and the intestinal tract. It is unique among glycosaminoglycans in that it is nonsulfated. Due to its abundant negative charges, HA can bind cations and absorb large amounts of water, acting as an osmotic buffer in the native ECM and forming hydrogel. Hence, hyaluronic acid may be an excellent alternative to other components of ECM, and thanks to its excellent water retention, hyaluronic acid provides the homeostasis of ECM hydration for tissues and joints and is responsible for resistance to the compression by physical force. In addition, hyaluronic acid is involved in permeability regulation between tissues, functions to induce a lubricant effect on friction in the joints, and acts as a carrier for providing nutrients to or removing wastes from avascular regions in the joint.
Taking advantage of its high water absorptivity and viscosity, accordingly, the naturally occurring hyaluronic acid itself or its derivatives (Glenn D. Prestwich, Jing-wen Kuo, Chemically-Modified HA for Therapy and Regenerative Medicine, Current Pharmaceutical Biotechnology, 9(4), 242-245 (2008)) have been applied to cosmetics or medically to the eyes (E. A. Balazs, M. I. Freeman, R. Kloti, G. Meyer-Schwickerath, F. Regnault, and D. B. Sweeney, “Hyaluronic acid and replacement of vitreous and aqueous humor”, Mod. Probl. Ophthalmol., 10, 3˜21 (1972)). In addition, hyaluronic acid finds various applications in the field of tissue regeneration and engineering, including the regeneration of cartilage and bones (A. H. Isdale, L. D. Hordon, H. A. Bird, and V. Wright, “Intra-articular hyaluronate (Healon): A dose-ranging study in rheumatoid arthritis and osteoarthritis”, J. Drug Dev., 4, 93˜99 (1991). M. Kawabata, M. Igarashi, R. Mikami, S. Ninomiya, and H. Oda, “Clinical evaluations of SLM-10 (sodium hyaluronate injection) in patients with osteoarthritis of the knee”, Yakuri to Chiryo, 21, 257˜283 (1993)), the reconstruction of the skin and soft tissues, and the resurfacing and plastic surgery of repressed tissues, for example, by directly injecting hyaluronic gel into the body.
Despite its useful applicability as a medical biomaterial, hyaluronic acid is limited in many of its uses because of its short half life in vivo. In fact, hyaluronic acid is too easily degraded in vivo, with a shorter half life than collagen. In bone and cartilage where lymph fluid is not secreted, it is probable that hyaluronic acid turnover occurs by metabolic degradation in situ concurrently with that of collagen and proteoglycans. In skin and joints, 20˜30% of hyaluronic acid is probably turned over by local metabolism, and the rest is removed by the lymphatic pathway. The tissue half-life of hyaluronic acid ranges from half a day to 2 or 3 days (J. R. E. Fraser, T. C. Laurent, and U. B. G. Laurent, Hyaluronan: its nature, distribution, functions, and turnover, J. Intern. Med., 242(1), 27-33 (1997)). Particularly, when used as an implant in a normal joint, hyaluronic acid is reported to have a half life of less than one day (T. C. Laurent, and J. R. E. Fraser, Hyaluronan, FASEB J., 6, 2397-2404 (1992)). The half life of hyaluronic acid is extended to 70 days in the eye where hyaluronic acid is not combined with other glycosaminoglycans (U. B. G. Laurent, and R. K. Reed, Turnover of hyaluronan in the tissue, Adv. Drug Delivery Rev., 7, 237-56 (1991)).
In spite of its great potential as a medical biomaterial, hyaluronic acid is currently limitedly used because of its fast degradability and short half life in vivo as well as the low mechanical properties of the natural polymer itself. For use in plastic surgery, hyaluronic acid gel must retain a desired mechanical strength for a long period of time in the body, and is usually prepared from hyaluronic acid with an ultrahigh molecular weight of 2,000˜3,000 kDa because it has a short half life.
Further, hyaluronic acid is required to be chemically modified and developed into derivatives that retain the hyaluronic acid structure but do not undergo the rapid degradation in vivo, so that they can be used as biomaterials for various clinical purposes. Many chemical modification attempts have been made on hyaluronic acid. For example, hyaluronic acid is crosslinked, is prepared into alkyl and benzylester derivatives, or modified with a coupling agent. Reviewing the research reports known thus far, hyaluronic acid derivatives developed by chemical modifications have proven suitable as medical polymers having mechanical and chemical properties for use in target tissues, organs and drug delivery systems. Some of them were shown to retain the hyaluronic acid's intrinsic biological functions in light of pharmaceutical functions. However, it is difficult to synthesize hyaluronic acid derivatives with a molecular weight of 1.5×106 Daltons or greater by chemical modification because such high-molecular weight polymers are likely to undergo intermolecular entanglement (Y. S. Soh, “Hyaluronic acid: properties and application”, Polymer (Korea), 12, (1988)).
Various solutions to the above-mentioned problems have been suggested. For example, a hyaluronic acid with a lower molecular weight may be prepared, or after the N-acetyl-D-glucosamine moiety is deacetylated with hydrazine, the resulting hyaluronic acid with an organic amine group may be chemically modified (V. Crescenzi, A. Francescangeli, D. Renier, D. Bellini, New cross-linked and sulfated derivatives of partially deacetylated hyaluronan: Synthesis and preliminary characterization, Biopolymers, Vol. 64, 86-94 (2002). S. Oerther, A-C Maurin, E. Payan, P. Hubert, F. Lapicque, N. Presle, J. Dexheimer, P. Netter, and F. Lapicque, High Interaction alginate-hyaluronate associations by hyaluronate deacetylation for the preparation of efficient biomaterials, Biopolymer, 54, 273-281 (2000)). In addition, the carboxyl group at position 6 of the beta-glucuronic acid, known as the target site of hyaluronidase, may be chemically substituted to prepare water-insoluble hyaluronic acid gels (Oh E J, Kang S W, Kim B S, Jiang G, Cho I H, Hahn S K., Control of the molecular degradation of hyaluronic acid hydrogels for tissue augmentation. J Biomed Mater Res A., 86(3):685-93 (2008) Hahn S K, Park J K, Tomimatsu T, Shimoboji T., Synthesis and degradation test of hyaluronic acid hydrogels. Int J Biol Macromol., 40(4), 374-80 (2007).). Further, chemical modifications may be made not only on the alcohol (—OH) groups within the repeating units, but also by disrupting the sugar ring structures, and a physical modification using the negatively charge on the carboxylic acid group has also been reported (V. Crescenzi, A. Francescangeli, D. Renier, D. Bellini, New hyaluronan chemical derivatives. Regioselectively C (6) oxidized products, Macromolecules, 34, 6367-6372 (2001)). However, chemical deacetylation, and chemical modification using a coupling agent, produces hyaluronic acid with a significantly reduced molecular weight, which results in weakening of the intrinsic mechanical property of hyaluronic acid (S. Oerther, A-C Maurin, E. Payan, P. Hubert, F. Lapicque, N. Presle, J. Dexheimer, P. Netter, and F. Lapicque, High Interaction alginate-hyaluronate associations by hyaluronate deacetylation for the preparation of efficient biomaterials, Biopolymer, 54, 273-281 (2000)). Furthermore, the deacetylation or modification may cause the separation of multivalent metals used for ionic crosslinking and the dissociation of introduced functional groups, which, together with degraded hyaluronic acid, are highly apt to exert cytotoxicity. In addition, hyaluronic acid with reduced mechanical strength is greatly limited in its use as a biomaterial in medical and industrial fields, and cannot be formulated into various types of biomaterial. Particularly when hydrogels, sheets, film, beads, or nanofibers are applied as tissue engineering scaffolds to regenerative medicine, their strengths and morphologies must be maintained until surrounding cells are introduced into the scaffolds so as to rapidly construct tissues. Otherwise, their morphologies may readily collapse, with a significant reduction in efficacy and engraftment to surrounding tissues. Therefore, in order to maintain the framework necessary of hyaluronic acid to act as a tissue engineering scaffold as well as serving as a carrier of cells, proteins, metals and drugs, and as a coating agent, the biodegradability of hyaluronic acid and its molecular weight must be possible to regulate.
On the other hand, deacetylation hydrolase of the N-acetyl-D-glucosamine moiety has been studied, mainly with chitin deacetylation hydrolase (CDA: E, C, 3.5.1.41), which catalyzes the conversion of chitin into chitosan. In 1936, Watanabe reported the likelihood of the existence of N-acetyl-D-glucosamine deacetylase in animal tissues, and S. Roseman first isolated the enzyme from an extract of E. coli strain K-12 in 1957. Subsequently, chitin deacetylation hydrolase has successfully been isolated from an extract of Mucor rouxii, and an extract of Bacillus cereus; fungi, such as Colletotrichum lindemuthianum, Colletotrichum lagenarium, and Rhizopus stolonifer; insect species; crustacea; and Encephalitozoon cuniculi, which is a protozoa. In addition, research results of the isolation and purification of chitin deacetylation hydrolase from Mucor rouxii, Absidia coerulea, and Aspergillus nidulans were reported. Isolation of cobalt-activated chitin deacetylation hydrolase (Cda2P) from Gongronella butleri and Saccharomyces cerevisiae, and chitin deacetylation hydroloase from Encephalitozoon cuniculi, Metarhizium anisopliae, and culture media of E. coli and Rhizopus oryzae was successful. In recent years, there have been reports on the isolation of chitin deacetylation hydrolase from Scopulariopsis brevicaulis, Mortierella sp. DY-52, Rhizopus circinans, and Vibrio cholera, but purification results thereof have not yet established.
In addition, since the finding that there is structural similarity between fungal chitin deacetylases and rhizobial nodulation proteins (NodB proteins), sequences of chitin deacetylases from fungi such as Mucor rouxii, Colletotrichum lindemuthianum, Saccharomyces cerevisiae, Gongronella butleri, and Rhizopus nigricans have been examined, but not yet reported.
As illustrated above, research into deacetylation hydrolase of polysaccharides has been centered around chitin deacetylation hydrolases, which, while not reactive to the monosaccharide N-acetyl-D-glucosamine, are able to act catalytically on a series of consecutive N-acetyl-D-glucosamine residues. Such chitin deacetylation hydrolase do not show enzymatic activity on the peptidoglycan N-acetylated heparin, and N-acetyl-galactosamine (Araki, Y. & Ito, E. (1975). A pathway of chitosan formation in Mucor rouxii, Eur. J. Biochem., 55, 71-78 (1975)), and cannot catalyze the deacetylation of the N-acetyl-D-glycosamine moiety of hyaluronic acid (Martinou A, Kafetzopoulos D, Bouriotis V., Chitin deacetylation by enzymatic means: monitoring of deacetylation process, Carbohyd. Res., 273, 235-242 (1995)). Like this, the chitin deacetylation hydrolase have been reported to have no enzymatic activity except for on N-acetyl-D-glucosamines of chitin and chitosan. That is, nowhere has a deacetylase selective for the acetyl group of the N-acetyl-D-glucosamine moiety of hyaluronic acid been reported in any previous documents.
There is therefore a need for research and development of a deacetylation hydrolase that can selectively deacetylate the N-acetyl-D-glucosamine moiety of hyaluronic acid.